Human Reserve Stem Cells Derived From Connective Tissues
Six populations of reserve stem cells derived from the connective tissues of skeletal muscle and dermis were used in these experiments. These populations were derived from fetal (female and male), mature (two female) and geriatric (female and male) donors. The protocols for harvesting human tissues have been approved by the Institutional Review Board at the Medical Center of Central Georgia, Macon, GA.
Cells derived from the connective tissues of skeletal muscle and dermis obtained from fetal and mature donors were purchased from Clonetics (San Diego, CA) and processed by differential plating and cryopreservation, yielding putative stem cells (Young et al., 1991, 1992a, 1999). In brief, the cells were transferred to plating medium-A (PM-A). PM-A consisted of 89% (v/v) Eagle's minimal essential medium with Earle's salts (EMEM, GIBCO BRL, Life Technologies, Grand Island, NY), 10% (v/v) pre-selected horse serum (lot numbers 17F-0218 [HS7] or 49F-0082 [HS4], Sigma Chemical Co., St. Louis, MO), and 1% (v/v) penicillin/streptomycin (10,000 U/ml penicillin and 10,000 mg/ml streptomycin, GIBCO), pH 7.4. Cells were incubated in a 95% air/5% CO2 humidified environment. After expansion, cells were released with 0.05% (w/v) trypsin (DIFCO, Becton-Dickinson Labware, Franklin Lakes, NJ) in Ca+2,Mg+2-free Dulbecco's phosphate buffered saline (GIBCO) containing 0.0744% (w/v) ethylenediamine tetraacetic acid (EDTA, Sigma), centrifuged at 100 × g for 20 min, and the supernatant aspirated. The cell pellet was resuspended in PM-A and the cell suspension cryopreserved by slow freezing for storage at −70 to −80°C in PM-A containing 7.5% (v/v) dimethyl sulfoxide (DMSO, Morton Thiokol, Danvers, MA) (Young et al., 1991). These cell lines were designated “CF-SkM1” (fetal female skeletal muscle connective tissue, CC-2561, lot number 14722, Clonetics), “CM-SkM1” (fetal male skeletal muscle connective tissue, CC-0231, lot number 6F0604, Clonetics), “NHDF1” (25-year-old female dermis, CC-0252, lot number 6F0600, Clonetics), and “NHDF2” (36-year-old female dermis, CC-0252, lot number 16280, Clonetics).
Geriatric cells were isolated from the endomysial, perimysial and epimysial connective tissue compartments associated with skeletal muscle specimens obtained from a 77-year-old female patient and a 67-year-old male patient following standard protocols for the isolation of mesenchymal stem cells (Lucas et al., 1995; Young et al., 1999). In brief, cells were liberated from the connective tissue compartment of skeletal muscle with collagenase (CLS-I, Worthington Biochemical Corp., Freehold, NJ) and dispase (Collaborative Biomedical Products, Bedford, MA). Single cell suspensions were obtained by sequential filtration through 90-μm and 20-μm Nitex (Tetco Inc., Elmsford, NY). Cells were seeded at 105 cells/1% (w/v) gelatin-coated (EM Sciences, Gibbstown, NJ) T-75 flasks (Falcon, Becton-Dickinson Labware) in PM-A and allowed to expand and differentiate before cryopreservation. Cells were incubated as before. After expansion, cells were released with trypsin, sieved as above to separate mononucleated cells from differentiated phenotypes (i.e., multinucleated myotubes, adipocyte colonies, cartilage nodules, bone nodules), and cryopreserved at −70 to −80°C in PM-A containing 7.5% (v/v) DMSO. Using the procedures outlined above, each subsequent cryopreservation step effectively removed more than 98% of contaminating fibroblasts and differentiated phenotypes from the stem cell preparation (Young et al., 1991). These cells were designated as “PAL2” and “PAL3”, respectively.
After initial harvest, further expansion of the cell lines through 30 cell doublings and more than 70 cell doublings was accomplished by repeated propagation and cryopreservation utilizing 1% gelatin coated flasks with plating medium-B (PM-B) (Young, 2000; Young et al., 1991, 1993, 1998b). PM-B consisted of 89% (v/v) Opti-MEM based medium (22600-050, GIBCO) containing 0.01 mM β-mercapto-ethanol (Sigma), 10% (v/v) horse serum (HS3, lot number 3M0338, BioWhittaker, Walkersville, MD), and 1% (v/v) antibiotic-antimycotic solution (10,000 U/ml penicillin, 10,000 μg/ml streptomycin, 25 μg/ml Amphotericin-B, GIBCO), pH 7.4. Cells were then propagated to 30 cell doublings and beyond, released with trypsin, and aliquoted for insulin/dexamethasone analyses and flow cytometric analyses. At each harvest, the cells were counted and the number of cells compared with the original plating densities. The number of cell doublings per passage was determined (Young, 2000; Young et al., 1991, 1993, 1995). Programmed senescence has been shown to occur at approximately 50–70 cell doublings (Hayflick's limit; Hayflick, 1965). The number of cell doublings was chosen so that one population would consist of cells at 30 cell doublings (less than Hayflick's limit). This population included both progenitor cells and pluripotent stem cells. Another population was chosen so that it would consist of cells beyond 70 cell doublings (at more than Hayflick's limit). As progenitor cells die around Hayflick's limit, the second population should be limited to pluripotent stem cells.
Cells were examined using an insulin/dexamethasone bioassay to determine the existence of progenitor or pluripotent stem cells within the populations examined (Young, 2000; Young et al., 1998a,1998b). In this bioassay, insulin accelerates the phenotypic expression of progenitor stem cells but has no effect on the induction of phenotypic expression in pluripotent stem cells. By contrast, dexamethasone induces lineage-commitment and expression in pluripotent stem cells, but does not alter phenotypic expression in progenitor stem cells (Young, 2000; Young et al., 1993, 1998a,1998b). Therefore, if progenitor cells alone are present in the culture there will be no difference in either the quality or quantity of phenotypes expressed upon treatment with insulin. Similar cultures will exhibit the expression of multiple phenotypes upon treatment with dexamethasone. If the culture is mixed, containing both progenitor and pluripotent cells, there will be a greater quality or quantity of phenotypes expressed upon treatment with dexamethasone compared with treatment with insulin. Thus, comparing the effects of treatment with insulin and dexamethasone permits the identification of specific types of progenitor and pluripotent stem cells within an unknown population of cells (Young, 2000; Young et al., 1998b, 1999).
Cells were processed as described previously (Young et al., 1999). In brief, aliquots of CM-SkM, CF-SkM, NHDF1, NHDF2, PAL#2, and PAL#3 cells were thawed and plated individually at 10,000 cells/well in 1% gelatin-coated 24-well plates (Corning, Corning, NY) utilizing PM-B. After 24 hr PM-B was removed and replaced with either control medium, insulin testing medium, or dexamethasone testing medium. Control medium consisted of 98% (v/v) Opti-MEM containing 0.01 mM β-mercapto-ethanol, 1% (v/v) HS3, and 1% antibiotic-antimycotic solution, pH 7.4. Insulin testing medium consisted of control medium containing 2 μg/ml insulin (Sigma), pH 7.4. Dexamethasone testing medium was composed of 98% Opti-MEM, 0.01 mM β-mercaptoethanol, 1% serum (HS3 or HS9, 90H-0701, Sigma) or FBS (fetal bovine serum, lot number 3000L, Atlanta Biologicals, Norcross, GA) and 1% antibiotic-antimycotic solution, pH 7.4. This solution was made 10−10, 10−9, 10−8, 10−7, or 10−6 M with respect to dexamethasone (Sigma) (Young, 2000; Young et al., 1995, 1998, 1999). Media were changed three times per week for 6 to 8 weeks. Cultures were viewed twice per week for changes in phenotypic expression and photographed.
The phenotypic expression of the cells was assayed morphologically. The morphological changes observed were identical to those previously noted in avian, mouse, rat, rabbit, and human progenitor and pluripotent mesenchymal stem cells after incubation with insulin or dexamethasone (Pate et al., 1993; Rogers et al., 1995; Young, 2000; Young et al., 1993, 1995, 1998a,1998b). Insulin induces a 1–5% expression in lineage-committed progenitor cells (Table 1) and dexamethasone induces a 6–10% expression in lineage-uncommitted cells (Tables 1 and 2). Phenotypes were expressed at all concentrations of dexamethasone from 10−10 to 10−6 M. Maximal expression of a particular phenotype, however, differed with respect to both dexamethasone concentration and time in culture.
Table 1. Induction of the expression of different mesodermal morphological phenotypes by insulin and dexamethasone in human stem cells at 30 cell doublingsa
| ||Insulin (2 μg/ml)||Dexamethasone (10−10 to 10−6 M)|
| IA4 + MF-20||−||−||−||−||−||−||++||++||++||++||++||++|
| Rd + PCMHi||+||+||+||+||+||+||++||++||++||++||++||++|
| SpM + Pk||−||−||−||−||−||−||++||++||++||++||++||++|
Table 2. CD marker expression profilea
| ||Prenatal donors||Postnatal donors|
|CM-SkM1||CF-SkM1||Mean ± SD||NHDF1||NHDF2||PAL3||PAL2||Mean ± SD|
When undergoing myogenesis, skeletal muscle recapitulates its developmental sequence. This sequence begins with single mononucleated stellate cells that stain intracellularly for myogenin (F5D, Developmental Studies Hybridoma Bank, DSHB; Wright et al., 1991), and then for sarcomeric myosin (MF-20, DSHB; Bader et al., 1982). Subsequently, bipolar cells that stain intracellularly for sarcomeric myosin appear. The bipolar cells then fuse to form multinucleated structures that stain intracellularly for sarcomeric myosin, fast-skeletal muscle myosin (MY-32, Sigma; Naumann and Pette, 1994), myosin heavy chain (ALD-58, DSHB; Shafiq et al., 1984), and human fast myosin (A4.74, DSHB; Webster et al., 1988). Skeletal structures undergoing myogenesis are further identified by their elongated structure, multiple nuclei, cross-striations, and spontaneous contractility (Pate et al., 1993; Rogers et al., 1995; Young, 2000; Young et al., 1992a, 1993, 1995, 1998a,1998b). Maximal expression of skeletal muscle markers was induced with 10−8 M dexamethasone within the first two weeks of culture.
A smooth muscle cell phenotype was identified by morphological criteria. This phenotype consisted of large polygonal cells containing intracellular stress fibers. The smooth muscle phenotype was defined in this study by immunocytochemical analysis using an antibody to smooth muscle alpha-actin (1A4, Sigma; Skalli et al., 1986). Maximal expression of smooth muscle alpha-actin was induced with 10−8 M dexamethasone within the first 2 weeks of culture.
A cardiac myocyte phenotype was identified by morphological criteria. This phenotype consisted of binucleated cells exhibiting centrally located nuclei, and cross-striations. The cardiac myocyte phenotype was defined in this study by immunochemical analysis using colabeling of antibodies for both smooth muscle alpha-actin (IA4) and sarcomeric myosin (MF-20) (Eisenberg et al., 1997; Eisenberg and Markwald, 1997). Maximal expression of smooth muscle alpha-actin colocalizing with sarcomeric myosin in binucleated cells was induced by treatment with 10−8 M dexamethasone within the first 2 weeks of culture.
Adipogenic cells were identified by morphologic criteria. They appeared as polygonal cells containing multiple intracellular refractile vesicles. Adipocytes were verified by the presence of intracellular vesicles containing saturated neutral lipid by means of histochemical staining with Sudan Black-B (Chroma-Gesellschaft, Roboz Surgical Co, Washington, DC; Young et al., 1993) and Oil Red-O (Sigma; Humason, 1972). Maximal expression of adipogenic markers was induced by treatment with 10−9 M dexamethasone within the first 2 weeks of culture.
Chondrogenic structures develop along a progressional sequence. Initially, stellate cells express intracellular staining for type-II collagen [collagen pro type-II (C11C1, DSHB; Holmdahl et al., 1986; Johnstone et al., 1998) and human-specific collagen type-II (II-4CII, ICN Biomedicals, Aurora, OH; Burgeson and Hollister, 1979; Kumagai et al., 1994)]. Intracellular staining for type-IX collagen (D1-9, DSHB; Ye et al., 1991) follows. The cells become circular, expressing refractile pericellular matrix halos, and aggregate into nodules. The pericellular matrix halos demonstrate extracellular staining for type-II collagen and type-IX collagen and histochemical staining for sulfated glycosaminoglycans using Alcian Blue at pH 1.0 (Chroma-Gesellschaft; Young et al., 1993, 1998a,1998b) and Perfix/Alcec Blue (Fisher Scientific Co., Norcross, GA/Aldrich Chemical Co., Milwaukee, WI). Loss of staining after preincubation with chondroitinase-AC and keratinase (Young, 2000; Young et al., 1993, 1998a,1998b, 1999) confirms the presence of glycosaminoglycans containing chondroitin sulfate and keratan sulfate within the pericellular matrix halos stained with Alcian Blue pH 1.0. Maximal expression of intracellular chondrogenic markers was induced by treatment with 10−7 M dexamethasone by the end of 2 weeks of culture. Cartilage nodule formation with extracellular staining characteristic of chondrogenic cells occurred by 4 weeks when cultures were treated with 10−7 M dexamethasone.
Putative osteogenic structures develop along a progressional sequence. Initially, stellate cells express intracellular staining for bone sialoprotein (WV1D1, DSHB; Kasugai et al., 1992) and osteopontin (MP111, DSHB; Gorski et al., 1990). The stellate cells then form circular cells that continue to exhibit intracellular staining for bone sialoprotein and osteopontin. The round cells aggregate to form a three-dimensional extracellular matrix. The extracellular matrix stains with antibodies to bone sialoprotein and osteopontin. It also exhibits histochemical staining for calcium phosphate using the von Kossa procedure (Silber Protein, Chroma-Gesellschaft; Young, 2000; Young et al., 1993, 1995, 1998a,1998b, 1999). The von Kossa procedure stains for divalent cations. The identity of calcium as the cation stained (rather than zinc or magnesium) was confirmed using preincubation with EGTA (a chelating agent specific for calcium ions) rather than EDTA (a chelating agent for divalent cations) (Young, 2000; Young et al., 1993, 1995, 1998a,1998b, 1999). Maximal expression of intracellular osteogenic markers was induced with 10−9 M dexamethasone by the end of 4 weeks of culture. Mineralized nodule formation with extracellular staining specific for osteogenic cells occurred by 6 weeks when cultures were incubated with 10−9 M dexamethasone. Fibroblasts were identified by their spindle-shaped or polygonal-shaped morphology. The fibrogenic phenotype was verified by immunocytochemical staining with antibodies to human fibroblast surface protein (1B10, Sigma; Ronnov-Jessen et al., 1992). Maximal expression of the fibrogenic marker was induced by treatment with 10−8 M dexamethasone after 2 weeks of culture.
Putative endothelial cells develop along a progressional sequence. Initially, stellate cells express intracellular staining for human-specific endothelial cell surface marker (P1H12, Accurate, Westbury, NY; Solovey et al., 1997), peripheral endothelial cell adhesion molecule, PECAM (P2B1, DSHB), vascular cell adhesion molecule, VCAM (P8B1, DSHB; Dittel et al., 1993), and E-selectin (P2H3, DSHB). The stellate cells then form cobblestone-shaped cells, which are present individually or in sheets. Maximal expression of endothelial markers was induced by treatment with 10−8 M dexamethasone for 2 weeks of culture.
Secondary antibodies consisted of biotinylated anti-sheep IgG (Vector), biotinylated anti-mouse IgG (Vector), or were contained within the Vecstatin ABC Kit (Vector). The tertiary probe consisted of avidin-HRP contained within the Vecstatin ABC Kit (Vector). The insoluble HRP substrates VIP Substrate Kit for Peroxidase (blue, Vector), DAB Substrate for Peroxidase (black, Vector), and AEC Staining Kit (red, Sigma) were used to visualize antibody binding. The different substrates were utilized to allow for multiple sequential staining of the same culture wells.
Aliquots of CM-SkM1, CF-SkM1, NHDF1, NHDF2, PAL3, and PAL2 cells at less than Hayflick's limit were thawed and seeded at 105 cells/1% gelatinized T-75 flasks in PM-B, and allowed to expand at 37°C in a 95% air/5% CO2 humidified environment. After expansion, cells were released with trypsin and resuspended in medium. The cells were then centrifuged and resuspended in wash buffer (Dulbecco's phosphate buffered saline without Ca+2, Mg+2 [Cellgro, MediaTech] supplemented with 1% FBS [HyClone] and 1% (w/v) sodium azide, NaN3 [Sigma]) at a concentration of 1 × 106 cells/ml. Cell viability was greater than 95% by the Trypan blue dye [GIBCO] exclusion technique (Young et al., 1991, 1993) and greater than 98% by the propidium iodide [Calbiochem-Novabiochem Corporation, La Jolla, CA] exclusion technique (Sasaki et al., 1987). One hundred microliters of cell preparation (1 × 105 cells) were stained with saturating concentrations of fluorescein isothiocyanate- (FITC), phycoerythrin- (PE), allophycocyanin (APC), or perdinin chlorophyll protein- (PerCP) conjugated CD3, CD4, CD8, CD11c, CD33, CD34, CD36, CD38, CD45, CD90, CD117, glycophorin-A, and HLA-II (DR), or isotype matched controls (Becton Dickinson, Inc. San Jose, CA). Briefly, cells were incubated in the dark for 30 min at 4°C. After incubation, cells were washed three times with wash buffer and resuspended in 0.5 ml of wash buffer. Flow cytometry was performed on a FACSort™ (Becton Dickinson) flow cytometer. Light scatter (Fig. 2) identified cells. Logarithmic fluorescence was evaluated (4 decade, 1,024 channel scale) on 10,000 gated events. Analysis was performed using Cellquest™ software (Becton Dickinson). The presence or absence of staining was determined by comparison with the appropriate isotype control. Gated events were scored for the presence of staining for a CD marker if more than 25% of the staining was above its isotype control. Percentages of cells per 10,000 gated events are shown in Table 3. A mean value above 10%-gated cells is considered positive for any given CD marker. Each cell line was run in triplicate, for a sample size of n = 3. Statistical analysis was performed on the pooled data from the six cell lines.
Table 3. CD cell surface markers on hematopoietic and neuronal cells
| ||CD3||CD4||CD8||CD11c||CD33||CD36||CD38||CD45||CD117||Gly-A DR-II|
|Natural killer cellsa||+||+||+||+|
|Myeloid progenitor cellsa||+||+||+||+|
|Some neuronal cellsb||+||+|